The present invention relates to methods and intermediates for chemical synthesis of polypeptides and proteins, and more particularly to methods and intermediates for chemically ligating a peptide fragment containing N-terminal N-methyl-cysteine (SEQ ID NO:1) with another peptide fragment having C-terminal thioester to generate a β-(methylamino)-thioester intermediate that spontaneously rearranges to form an amide bond. Furthermore, the invention relates to methods of converting N-methyl-thiazolidine to N-methyl-cysteine (SEQ ID NO:1) of polypeptides and proteins. The invention also relates to methods of synthesizing peptide-thioester from peptide-acid fluoride.
Several techniques for chemically synthesizing proteins have been developed. See, e.g., Stewart, J. M. et al., Solid Phase Peptide Synthesis (Pierce Chemical Co., 2d ed., 1984), and Bodanszky, M. et al., The Practice of Peptide Synthesis (Springer-Verlag, 1984). Among them, native chemical ligation has proven to be one of most useful methods for chemically generating native proteins. However, native chemical ligation is only suitable for the synthesis of polypeptides and proteins having cysteine residue(s), which can be used as the connection point(s) for ligating peptide fragments to form the final target polypeptides and proteins.
To produce polypeptide and protein analogs and derivatives which have improved chemical and biological properties, incorporation of unnatural amino acid residue(s) into specific position(s) inside the polypeptides and proteins is sometimes required. Such analogs and derivatives can have improved chemical stability, improved enzymatic stability, prolonged duration of action in vivo, and enhanced biological activities. One class of such unnatural amino acids is the N-methyl amino acids. The N-methyl amino acids can impose conformational constraint on peptide backbone, block hydrogen bonding sites and potentially protect the peptide bonds against enzymatic cleavage (see, e.g., Haviv, F. et al., J. Med. Chem., 1993, 36:363-369; Failie, D. P., et al., Curr. Med. Chem., 1995, 2:654-686; Miller, S. M., et al., Drug Dev. Res., 1995, 35:20-32; and Schmidt, R., et al., Int. J. Pept. Protein Res., 1995, 46:47-55). N-methyl cysteine (SEQ ID NO:1) is one member of this class of unnatural amino acids. To generate biologically more active, enzymatically more stable protein analogs and derivatives, there is a need to develop a new chemical method to incorporate N-methyl-cysteine (SEQ ID NO:1) residue in any desired position inside of proteins.